The TGFβ Pathway
TGFβs are secreted polypeptides that are critical inhibitors of epithelial growth, immune and hematopoietic functions and activators of connective tissue growth (reviewed by Massague, J. and Y. G. Chen, Genes Dev. 14:627–44, 2000). TGFβ1, 2, and 3 are three members of the large TGFβ superfamily that also includes the bone morphogenetic proteins, the growth and differentiation factors (GDFs), nodal-related proteins and activins. TGFβs signal by activating two receptor serine/threonine protein kinases, the Type I and Type II receptors. The receptors phosphorylate Smad2 and Smad3 which form a heteromeric complex with Smad4 and translocate into the nucleus. The phosphorylated Smad complex (Smad-P) regulates transcription by interactions with several DNA binding proteins, transcriptional co-activator proteins and transcriptional repressor proteins. The signal is terminated by multi-ubiquitination of Smad and proteasome-mediated degradation (Lo, R. S. and J. Massague, Nat. Cell. Biol. 1:472–478, 1999).
The Smad proteins are key components of TGFββ signal transduction, carrying the signal into the nucleus and generating a diverse set of tissue-specific responses. Smad proteins have an N-terminal globular domain with DNA binding activity, a central linker region with regulatory sites, and a C-terminal globular domain with transcriptional regulatory activity (Liu, F., et al., Genes Dev. 11:3157–67, 1997; Liu, F., et al., Nature 381:620–3, 1996). Both the N-terminal and C-terminal domain structures have been solved by X-ray crystallography (Shi, Y., et al., Cell 94:585–94, 1998; Shi, Y., et al., Nature 388:87–93, 1997). The DNA binding activity is associated with a hairpin loop in the N-terminal domain. The DNA binding activity is probably not the basis of the selectivity since all Smads recognize the same sequence, CAGAC (Shi, Y., et al., supra, 1998). The DNA-binding activity of Smads is also low-affinity and probably plays a role only in the context of Smad association with other DNA binding proteins such as FAST, Mixer, Milk, AP-1, TFE-3, and AML (Chen, X., E., et al., Nature 389:85–9, 1997; Germain, S., et al., Genes Dev. 14:435–51, 2000; Zhang, Y., et al., Nature 394:909–13, 1998; Hua, X., et al., Proc. Natl. Acad. Sci. USA 96:13130–5, 1999; Pardali, E., et al., J. Biol. Chem. 275:3552–60, 2000). The specific interactions of Smad and the Type I receptor are dictated by the L3 loop in Smad and the L4,5 loop in the Type I receptor (Lo, R. S., et al., EMBO J. 17:996–1005, 1998). A specific alpha helical structure in the Smad C-terminal domain, αhelix-2, specifies the binding of Smad to the DNA binding protein FAST (Chen, Y. G., et al., Genes Dev. 12:2144–52, 1998). Smad-P complexes also recruit the transcriptional co-activators p300/CBP and the transcriptional co-repressors TGIF, c-Ski and Sno-N to promoters bound by Smad-DNA binding protein complexes. p300/CBP has intrinsic histone acetyltransferase activity that can facilitate transcription; the co-repressors recruit histone deacetylases (Wotton, D., et al., Cell 97:29–39, 1999; Luo, K., et al., Genes Dev. 13:2196–206, 1999; Janknecht, R., et al., Genes Dev. 12:2114–9, 1998; de Caestecker, M. P., et al., J. Biol. Chem. 275:2115–22, 2000). Smad7 is an antagonistic Smad that inhibits Smad2 or 3 activation by binding to TGFβ Type I receptors (Souchelnytskyi, S., et al., J. Biol. Chem. 273:25364–70, 1998). A second antagonistic Smad, Smad6, regulates BMP signaling by competing with Smad4 to bind phosphorylated Smad1 (Hata, A., et al., Genes Dev. 12:186–97, 1998). Smad7 expression is increased by TGFβ and by other signals that negatively regulate TGFβ signaling, e.g., interferon γ, TNFα and interleukin 1β (Bitzer, M., et al., Genes Dev. 14:187–97, 2000).
Although Smad2 and Smad3 are both involved in mediating the response to TGFβ, they exhibit several functional differences. The N-terminal domain of Smad2 contains a short additional sequence that disrupts the DNA binding of the Smad2 N-terminal domain; the MH1 domain of Smad 3 can bind DNA. Smad2 interacts with FAST2 to activate transcription of the goosecoid promoter but Smad3 interaction with FAST2 suppresses transcription (Labbe, E., et al., Mol. Cell 2:109–20, 1998). Loss-of-function mutations in mice also exhibit different phenotypes. The unphosphorylated, cytoplasmic Smad proteins are also regulated by protein—protein interactions. SARA is a binding protein for Smad2 (but not Smad1) that facilitates phosphorylation of these Smads by the Type I receptor (Tsukazaki, T., et al., Cell 95:779–91, 1998). The cytoplasmic localization of Smad 2, 3, and 4 in epithelial cells may be due to association of these Smads with microtubules (Dong, C., et al., Mol. Cell 5:27–34, 2000).
TGFβ and Disease
There is an extensive literature on the role of TGFβ in a broad variety of human diseases including cancer (Akhurst, R. J. and A. Balmain, J. Pathol. 187:82–90, 1999; de Caestecker, M. P., et al., J. Natl. Cancer Inst. 92:1388–402, 2000; Taipale, J., et al., Adv. Cancer Res. 75:87–134, 1998), immunoregulation (Letterio, J. J. and A. B. Roberts, Annu. Rev. Immunol. 16:137–61, 1998), wound healing and tissue repair (Grande, J. P., Proc. Soc. Exp. Biol. Med. 214:27–40, 1997). As discussed by Akhurst and Balmain in their recent review, TGFβ has been implicated in tumor suppressor functions through its growth inhibitory properties on most epithelial and hematopoietic cell types. Loss-of-function mutations in TGFβ receptors and Smads allow cells to escape the grow-inhibitory functions of TGFβ. More indirect mechanisms, such as elevated mdm2, have also been proposed as ways that cells escape negative growth control (Sun, P., et al., Science 282:2270–2, p53-independent role of MDM2 in TGF-beta1 resistance.). And yet, as emphasized by Akhurst and Balmain, a more frequent role of TGFβ in cancer is to facilitate the progression and spread of tumor cells. Overexpression of TGFβ has been reported in advanced mouse and human carcinomas. The tumor-derived TGFβ can aid tumorigenicity by direct actions on the cancer cell, by induction of angiogenesis, by local or systemic immunosuppression, and by alterations of stromal tissue that facilitate invasiveness (Akhurst, R. J. and A. Balmain, supra, 1999).
Non-Smad Proteins Involved in TGF-β Signaling
Although the focus of the proposed screen disclosed below is on the critical Smad component of TGFβ signaling, it should be noted that there are other independent of the Smads. The role of these other pathways will be important to future design of combinatorial drug strategies for inhibition of TGFβ signaling, one component of which will be the Smad inhibitors to be identified by the proposed screens.
There are at least four different examples that illustrate how TGFβ mediated signal transduction processes are more complex than simple activation of Smads: (a) Negative regulation of Smad function by MAPK/ERK, (b) Positive regulation of Smad activation by MAPKK1, (c) Cooperative signaling initiated by TGFβ of both Smad and MAPK pathway activation, and (d) Smad-independent signaling by TGFβ.
One mechanism that is important to generating tissue-specific responses is cross-talk between the Smad pathway and other signaling pathways. Nuclear translocation of Smads is negatively regulated by MAPK phosphorylation of the Smad linker region (Kretzschmar, M., et al., Genes Dev. 13:804–16, 1999). Intracellular calcium levels can also negatively impact TGFβ signaling through Ca++-calmodulin dependent protein kinase II. Cam kinase II phosphorylates Smad2 at several sites (serines 110, 240 and 260) in response to activation of several growth factor receptor signaling pathways including the HER2 oncoprotein (Wicks, S. J., et al., Mol. Cell. Biol. 20:8103–11, 2000).
There are not yet many examples of Smads being used by other signaling pathways, but MEKK-1can activate Smad2 function in endothelial cells (Brown, J. D., et al., J. Biol. Chem. 274:8797–805, 1999). This result raises the possibility that Smads may be utilized by non-TGFβ signal transduction pathways.
There is evidence that TGFβ activates MAPK pathways, including the MKK4-JNK pathway in a human fibrosarcoma cell line (Hocevar, B. A., et al., EMBO J. 18:1345–56, 1999) and in the mink lung epithelial cell line Mv1Lu (Engel, M. E., et al., J. Biol. Chem. 274:37413–20, 1999). Although the precise molecular mechanism of MAPK-pathway activation by TGFβ receptors is not clear at this time, MAPKs can activate AP-1 and ATF2 transcription factors that act in concert, perhaps synergistically, with activated Smad-P at specific target promoters. Hocevar and colleagues report that inhibition of the MKK4-JNK pathway blocks TGFβ induction of fibronectin while having little effect on TGFβ induction of PAI-1. They propose that this specificity may be due to the requirement for c-jun-ATF2 heterodimers at the fibronectin promoter but not at the PAI-1 promoter (Hocevar, B. A., et al., supra, 1999).
Mulder and colleagues have reported on the rapid activation of Ras during TGFβ upregulation of Cdk inhibitors (p27Kip1 and p21Cip1). They showed that dominant negative Ras expression or the ERK kinase (MEK1) inhibitor PD98059 reduced phosphorylation of Smad1 by TGFβ stimulation in intestinal epithelial cells, and also inhibited the response of the 3TP-luciferase reporter gene to TGFβ (Yue, J., et al., Oncogene 18:2033–7, 1999). More recently, Mulder and Yue have reported the involvement of Ras, MKK4 and MEK1 in the formation of a Fra-2/JunD complex at an Ap-1 site in the TGFβ1 promoter. The Fra-2/JunD complex forms and activates TGFβ1 expression in response to TGFβ3 on intestinal epithelial cells (Yue, J. and K. M. Mulder, J. Biol. Chem. 275:30765–73, 2000). The activation of TGFβ1 expression was blocked by dominant negative mutations in Smad3 and Smad4, but the mechanism of this interference was not clear in that Smad3 and Smad4 were not detected in the transcriptional complex at the AP1 site formed in response to TGFβ3 (Yue, J. and K. M. Mulder, supra, 2000). In addition to MAPK involvement in TGFβ responses, PI3-kinase has been implicated in responses to TGFβ through the use of the PI3-kinase inhibitor Iy294002 or expression of dominant negative forms of PI3-kinase in rat hepatocytes. Peron and colleagues propose that EGF potentiates TGFβ signaling in rat hepatocytes by stimulation of AP1 through PI3-kinase and interaction of AP1 with Smad3 (Peron, P., et al., J. Biol. Chem. 27:27, 2000).
Up until recently, studies in genetic models systems such as Drosophila had emphasized the roles of the Smad-related genes in pathways activated by the TGFβ superfamily ligands such as dpp. However a recent study has found that a p38 MAPK in Drosophila (D-p38b) can alter the same phenotypes as dpp during Drosophila development. Dominant-negative forms of D-p38b and the p38 inhibitor SB203580 caused dpp-like phenotypes in Drosophila wing development and, importantly, suppressed mutant phenotypes caused by overexpression of an activated dpp receptor (Adachi-Yamada, T., et al., Mol. Cell. Biol. 19:2322–9, 1999). These results are consistent with D-p38b functioning in a positive response to Drosophila dpp signaling, perhaps cooperatively with Mad and Medea, the Drosophila Smads.
An example of a Smad-independent mechanism is the recent report that the activated TGFβ receptor directly phosphorylates protein phosphatase2A-Bα. This leads to association of the three PP2A subunits with p70s6k, dephosphorylation and inactivation of p70s6k, inhibition of translation of specific mRNAs and cell cycle arrest (Petritsch, C., et al., Genes Dev. 14:3093–101, 2000). In mammary epithelial cells, this translational mechanism provides a Smad-independent G1 cell cycle arrest in response to TGFβ whereas in other cell lines and in murine embryonic fibroblasts TGF-β does not inhibit p70s6k and Smad signaling is both necessary and sufficient for G1 arrest (Patritsch, C., et al., supra, 2000). This cell-type specificity appears to depend on whether the Bα subunit of PP2A is expressed in the cells.
Oncogenic Ras causes mammary epithelial cells to respond to TGFβ so as to enhance their invasive and metastatic potential instead of being growth inhibited (Oft, M., et al., Genes Dev. 10:2462–77, 1996; Oft, M., et al., Curr. Biol. 8:1243–52, 1998). Downward and colleagues reported a similar phenomenon in MDCK epithelial cells. Activated Raf increased the secretion of TGFβ, which is growth inhibitory to the parental MDCK cells. Cells with activated Raf were refractory to the growth inhibitory effects of TGFβ but retained TGFβ-dependent invasiveness (Lehmann, K., et al., Genes Dev. 14:2610–22, 2000). Raf-expressing cells lost their apoptotic responses to both TGFβ and TNF-α but retained TGF-β induced activation and nuclear translocation of Smad2, 3 and 4. Loss of the apoptotic response occurred more rapidly, within 8–24 hours, whereas loss of all growth inhibitory responses to TGFβ occurred after several weeks of Raf activation, coincident with the epithelial-mesenchymal transition in these cells (Lehmann, K., et al., supra, 2000). Iglesias and colleagues studied the effects of activated Ras on TGFβ signaling in murine keratinocytes (Iglesias, M., et al., Oncogene 19:4134–45, 2000). Similar to earlier reports from Mulder and colleagues of rapid activation of Ras and MAPK by TGFβ, Iglesias and colleagues observed maximal levels of Ras-GTP two minutes after TGFβ stimulation and nuclear localization of ERK approximately 30 minutes after TGFβ stimulation. In the Ras transformed keratinocytes, dominant negative Smad4 did not alter the TGFβ induction of p21Cip1, but the MEK inhibitor PD098059 did block this response to TGFβ. Furthermore, they reported that the dominant negative Smad4 led to hyper activation of Ras/Erk signaling, increased levels of urokinase secretion and more poorly differentiated carcinomas (Iglesias, M., et al., supra, 2000). Additional evidence for the complex relationship between oncogenic Ras and TGFβ signaling comes from analysis of human prostate cancer cells (Park, B. J., et al, Cancer Res. 60:3031–8, 2000). TGFβ stimulates the growth of the prostate cancer cell line TSU-Pr1 but in the presence of PD098059, TGFβ is growth inhibitory. The mitogenic effect of TGFβ was not inhibited by expression of dominant negative Smad2, Smad3 or Smad4 (Park, B. J., et al, supra, 2000). A recent report on the role of TGFβ in the epithelial to mesenchymal transition in tumor progression has identified another interesting pathway. Moses and colleagues report that TGFβ rapidly activates RhoA in a process that is not blocked by dominant-negative Smad3 or Smad7 and that causes induction of stress fibers and mesenchymal characteristics in mammary epithelial cells (Bhowmick, N. A., et al., Mol. Biol. Cell. 12:27–36, 2001).
Smad-P Protein—Protein Interactions
As the previous section illustrates, there are likely to be several pathways involved in TGFβ signaling. Some of these, such as the MAPKs and PI3 kinase are clearly also very important in cell responses to factors acting on receptor tyrosine kinases such as EGF, PDGF and the FGFs. There are already chemical inhibitors of some of these pathways and much work still being done to discover new pharmacological inhibitors of these pathways. In contrast, there is as yet, no pharmacological inhibitor specific to the Smad pathway.